Core Binding Factor.
Core binding factors (CBFs) are heterodimeric transcription factors consisting of a DNA-binding CBFα subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFβ subunit. Homozygous disruption of Runx1 or Cbfb in mice results in mid-gestation embryonic lethality and a profound block in definitive hematopoiesis (Okuda, van Deursen et al. 1996; Sasaki, Yagi et al. 1996; Wang, Stacy et al. 1996; Wang, Stacy et al. 1996), establishing these two proteins as essential for proper hematopoiesis. The embryos lack definitive hematopoietic progenitors, enucleated erythrocytes, and mature myeloid lineage cells because of the failure of hematopoietic stem cells to emerge from the endosteum at day E10.5. Runx1 deficient embryos lack any transplantable hematopoietic stem cell (HSC) activity, the lineage from which all differentiated lineages develop with the exception of erythrocytes, establishing CBF as a critical regulator of hematopoietic stem cell function (Cai, de Bruijn et al. 2000).
Studies in mice of dosage effects of Runx1 and Cbfb on hematopoiesis show significant effects with even modest changes in the level of either protein. Two-fold changes in Runx1 dosage affect timing of hematopoietic stem cell emergence in the embryo and the numbers of committed progenitors in the embryo and adult (Cai, de Bruijn et al. 2000; North, de Bruijn et al. 2002; Lacaud, Kouskoff et al. 2004). These studies suggest that the dosage of Runx1 affects the balance between hematopoietic stem cells and committed progenitors in the embryo. Consistent with this, alteration of RUNX dosage in humans is associated with disease. The familial platelet disorder with a propensity to develop AML (FPD/AML) syndrome is an inherited disease displaying altered hematopoiesis (thrombocytopenia and impaired platelet function) and a high risk of developing acute myeloid leukemia (AML) (Ho, Otterud et al. 1996; Song, Sullivan et al. 1999). These patients have loss-of-function mutations in RUNX1. In addition, 3-5% of sporadic leukemias display loss-of-function mutations in RUNX1 (Osato, Yanagida et al. 2001).
Alteration of Cbfb dosage also shows significant defects in hematopoietic compartments. A recent study showed that a 3-fold reduction in Cbfb dosage resulted in a reduction in the number mature thymocytes (T cells) and a 6-fold reduction resulted in no thymocytes (Talebian, Li et al. 2007). It is clear that the dosages of both Runx1 or Cbfb are critical for normal hematopoiesis and regulation of HSCs.
Structural and Functional Characterization of CBF.
The structure of the heterodimerization domain of CBFβ was first determined using NMR spectroscopy by our lab (Huang, Peng et al. 1999) and others (Goger, Gupta et al. 1999). The CBFβ heterodimerization domain has a novel α/β fold composed of a central six-stranded highly-twisted β-sheet surrounded by four α-helices and one 310 helix. NMR chemical shift perturbation analysis was used to map the binding site for the Runx1 Runt domain on CBFα (Huang, Peng et al. 1999). Based on this data, we carried out extensive Ala mutagenesis on CBFβ to identify the energetic hotspots for binding (Tang, Shi et al. 2000). Subsequent crystal structures of ternary complexes (see below) confirmed the importance of this region for interaction with Runx1.
We and others also determined the structure of the DNA and CBFβ binding domain of Runx1, termed the Runt domain, using NMR (Berardi, Sun et al. 1999; Nagata, Gupta et al. 1999). Structural studies of the Runt domain were carried out using Runt domain-DNA complexes because of the poor behavior of the isolated proteins in solution (Berardi, Sun et al. 1999; Nagata, Gupta et al. 1999; Perez-Alvarado, Munnerlyn et al. 2000). The Runt domain is a β-sandwich protein with no α-helical content, as was predicted from an earlier circular dichroism (CD) study (Crute, Lewis et al. 1996). The fold identified for the Runt domain belongs to the classic immunoglobulin (Ig) fold, specifically the s-type Ig fold best exemplified by the structure of the fibronectin repeat element. Interestingly, this fold has been identified in the DNA binding domains of a number of other critical mammalian transcription factors including NF-κB, NFAT, p53, STAT-1, and the T-domain. Thus, the Runt domain belongs to a family of structurally-related s-type Ig fold DNA-binding domains. For all of these structurally-related proteins, binding to DNA is mediated by the loop regions that connect the various β-strands. As was done for CBFβ, we carried out a chemical shift perturbation mapping study to identify the CBFα binding site on the Runt domain (Tang, Crute et al. 2000). Based on this data, we carried out extensive Ala mutagenesis to identify the energetic hotspots for CBFβ binding on the Runt domain (Zhang, Li et al. 2003). We also carried out an extensive Ala mutagenesis study to identify the energetic hotspots for DNA binding (Li, Yan et al. 2003).
Crystal structures of ternary complexes containing CBFβ, the Runt domain, and DNA have been determined using X-ray crystallography (Bravo, Li et al. 2001; Tahirov, Inoue-Bungo et al. 2001). These structures have provided a wealth of detailed information on the interaction between the Runt domain and DNA as well as between the Runt domain and CBFβ. As seen in all the other Ig-fold DNA-binding proteins mentioned above, the Runt domain makes contacts in both the major and minor grooves of the DNA using loops extending from one end of the barrel. These structures also clearly show the location and positioning of the CBFβ subunit. As predicted based on biochemical experiments, CBFβ does not make any direct contacts to the DNA, but modulates the Runt domain's binding affinity indirectly, i.e. allosterically.
Crystal structures of the Runx1 Runt domain alone have also been determined (Backstrom, Wolf-Watz et al. 2002; Bartfeld, Shimon et al. 2002). A previous NMR study of the isolated Runt domain alone was able to characterize the β-barrel, but limited solubility and extensive exchange broadening for resonances of the residues in the DNA-binding loops limited the structural characterization (Perez-Alvarado, Munnerlyn et al. 2000). With the addition of a high-resolution structure of the isolated Runt domain, detailed structural information is available for this domain alone, in complex with CBFβ or DNA, and in a ternary complex with both CBFβ and DNA. This makes possible comparisons among the structures to identify structural changes in the Runt domain that occur upon binding. Both CBFβ and DNA induce very similar changes in the structure of the Runt domain (Backstrom, Wolf-Watz et al. 2002; Bartfeld, Shimon et al. 2002), arguing for the concept that the CBFβ subunit does indeed stabilize the DNA-binding conformation of the Runt domain. Indeed, we have shown by NMR relaxation studies that CBFβ alters an existing conformational equilibrium in the Runt domain which explains how this allosteric regulation is achieved (Yan, Liu et al. 2004).
Inv(16) Leukemia.
Two of the four genes encoding CBF subunits are proto-oncogenes commonly activated in human leukemias. The RUNX1 subunit is encoded by the acute myeloid leukemia 1 (AML1) or RUNX1 gene which is disrupted by a variety of chromosomal translocations (Look 1997), all of which are associated with myeloid and lymphocytic leukemias. The gene encoding CBFβ (CBFB) is disrupted by the pericentric inversion of chromosome 16 [inv(16)(p13q22)], and less frequently by the t(16;16)(p13q22), associated with 100% of AML-M4Eo subtype (Liu, Tarle et al. 1993), resulting in a fusion protein containing most of the CBFβ protein (N-terminal 165 amino acids) fused to the coiled-coil tail region of smooth muscle myosin heavy chain (SMMHC) (see FIG. 1). The inv(16) is associated with ˜12% of de novo acute myeloid leukemias in humans (Look 1997). The CBFβ-SMMHC fusion protein acts as a dominant repressor of CBF function (Liu, Tarle et al. 1993), binding RUNX1 and dysregulating the expression of multiple genes required for normal hematopoiesis.
Mice heterozygous for a knocked-in Cbfb-MYH11 allele displayed a very similar phenotype to that seen for the complete knockout of Runx1 or CBFβ, namely lethality at embryonic day 12 accompanied by hemorrhaging and a severe block in definitive hematopoiesis (Castilla, Wijmenga et al. 1996). Heterozygous embryos expressing one copy of CBFβ-SMMHC and one copy of CBFβ displayed a very similar phenotype to that seen for the complete knockout of Runx1 or CBFβ, namely lethality at day 12 with severe hemorrhages and a complete block in hematopoietic development. Further analysis showed that the presence of CBFβ-SMMHC specifically blocks maturation of lymphoid and myeloid lineages. These data clearly establish CBFβ-SMMHC as a dominant negative inhibitor of CBF function.
Previous studies have shown that CBFβ-SMMHC is necessary but not sufficient to cause leukemia in mouse models. In studies carried out by Dr. Lucio Castilla, chimeric mice generated from CbfbCbfb-MYH11/+ embryonic stem cells were shown to be highly predisposed to the development of AML upon treatment with chemical (ENU) or retroviral mutagenesis (Castilla, Garrett et al. 1999) (Castilla, Perrat et al. 2004). The observed morphology of these mice closely mimics that seen for patients with AMLM4Eo. A more recent study in Dr. Castilla's lab used a conditional CbfbCbfb-MYH11/+ knock in mouse to demonstrate that activation of CBFβ-SMMHC expression in bone marrow induces accumulation of myeloid progenitors that transform to full blown leukemia in 3 to 5 months (Kuo, Landrette et al. 2006). All these studies clearly establish CBFβ-SMMHC as essential for inv(16) leukemogenesis and argue strongly that targeted inhibition of this fusion protein would have therapeutic value.
In patients with inv(16) leukemia, leukemic cells frequently present additional mutations that could synergize in leukemogenesis. The most common mutations are activating mutations in the receptor tyrosine kinases c-Kit and FLT3 (Reilly 2005). This agrees well with the (at least) “two-hit hypothesis” (Dash and Gilliland 2001; Gilliland and Tallman 2002) that postulates that leukemia requires a combination of a mutation that blocks differentiation and a second that enhances proliferation. Hits that block differentiation often involve transcription factors such as the creation of the CBFβ-SMMHC fusion protein, whereas those that enhance proliferation often involve mutations that create a ligand-independent activated receptor tyrosine kinase or the associated RAS pathway. In support of this view, a recent study showed cooperativity between inv(16) and the FLT3 activated form FLT3ITD (Kim, Klug Blood 2008). Interestingly, molecular analysis of human AML samples at diagnosis and relapse indicate that these secondary mutations are not always present at relapse, strongly suggesting that while inv(16) may occur in the HSCs, the FLT3-ITD mutation may arise in later progenitors.
Mechanism of CBFβ-SMMHC Mediated Leukemogenesis.
Expression of CBFβ-SMMHC in cultured cells results in altered cellular localization of Runx1. Normally, Runx1 shows nuclear localization, but when co-expressed with CBFβ-SMMHC, both Runx1 and CBFβ-SMMHC are found predominantly in the cytoplasm (Lu, Maruyama et al. 1995; Liu, Wijmenga et al. 1996; Adya, Stacy et al. 1998; Kanno, Kanno et al. 1998). These results form the basis for one of the proposed mechanisms of the dominant negative activity of CBFβ-SMMHC, namely that CBFβ-SMMHC sequesters Runx1 in the cytoplasm thereby preventing it from reaching the nucleus (reviewed in (Shigesada, van de Sluis et al. 2004)). The C-terminal tail of CBFβ-SMMHC which includes an assembly competence domain (ACD) has been shown by co-immunoprecipitation to bind to a number of proteins involved in transcriptional repression including mSIN3 and HDACs (Lutterbach, Hou et al. 1999; Durst, Lutterbach et al. 2003). Based on these results, a second model for the dominant negative activity of CBFβ-SMMHC has been proposed in which CBFβ-SMMHC acts at the level of the promoter as a transcriptional repressor by means of recruitment of specific co-repressors (reviewed in (Hiebert, Lutterbach et al. 2001)).
Importantly, neither Runx1+/− nor Cbfb+/− mice exhibit the dramatic hematopoietic defects associated with the CBFB-MYH11 knock-in allele (Castilla, Wijmenga et al. 1996; Okuda, van Deursen et al. 1996; Sasaki, Yagi et al. 1996; Wang, Stacy et al. 1996; Wang, Stacy et al. 1996). Neither of the two models mentioned above explained how more than half of the normal Runx1-CBFβ activity is lost. Our hypothesis was that CBFβ-SMMHC inactivated more than 50% of Runx1-CBFβ function because of its altered affinity for Runx1. Using isothermal titration calorimetry, we showed that CBFβ-SMMHC binds ˜10-fold more tightly to the Runx1 Runt domain than does wildtype CBFβ□(Lukasik, Zhang et al. 2002). NMR studies of a complex between a functional CBFβ-SMMHC domain and the Runt domain show that the Runt domain in this complex is contacting both the CBFβ portion of the protein and a specific region in the SMMHC domain. These results demonstrate that the increased affinity of CBFβ-SMMHC for Runx1 contributes to the disruption of normal hematopoiesis.
More recently, we have also shown that CBFβ-SMMHC inhibits the DNA binding of the Runt domain, providing yet another layer of inhibition of Runx activity. Consistent with this, previous studies have shown decreased RUNX binding to the MPO promoter (Cao, Britos-Bray et al. 1997) and to the INK4b promoter (Markus, Garin et al. 2007) in the presence of CBFβ-SMMHC. All of the functional studies clearly establish that the binding of CBFβ-SMMHC to the Runt domain of RUNX1 is essential for its function and therefore establish this as an appropriate target for inhibition.
Role of Inv(16) in Leukemia Stem Cell Properties.
Patients with inv(16) AML usually undergo aggressive chemotherapy regimes involving cytotoxic drugs such as Ara-C and anthramycin. This treatment is better tolerated by young patients showing a 5 year overall survival of 45% to 65% (Ravandi, Burnett et al. 2007; Pulsoni, Iacobelli et al. 2008). However, most patients are older and the 5 year overall survival for patients older than 60 years old is about 20% (Farag, Archer et al. 2006). These data clearly indicate targeted therapies that can improve the therapeutic response for inv(16) AML patients are essential.
Emerging literature suggests that inability to cure cancers with current therapies, including cytotoxic chemotherapy, kinase inhibitors, or monoclonal antibodies, may be attributed to a population of so-called cancer stem cells or cancer initiating cells that are resistant to treatment, are quiescent, have long term self-renewal potential, and can fully recapitulate tumor phenotype at time of relapse. Inv(16) AML is a good example of this failure because inv(16) patients invariably show, at time of relapse, the inv(16) rearrangement, although other mutations detected at diagnosis (RAS, FLT3ITD or KIT) may or may not be detected at relapse (Nakano, Kiyoi et al. 1999; Kottaridis, Gale et al. 2002; Shih, Liang et al. 2008).
Because of the critical role of CBFβ and RUNX1 in regulating hematopoietic stem cells, dysregulation of this pathway by CBFβ-SMMHC (or other CBF fusion proteins such as AML1-ETO or TEL-AML1), will lead to the aforementioned cancer stem cell properties such as long term self renewal potential. In addition, microarray analysis of CBF leukemias indicates alteration of the levels of proteins involved in DNA repair (Alcalay, Orleth et al. 2001; Xu, Li et al. 2007), leading to enhanced mutagenesis rates and a likely increase in the rate of acquisition of secondary mutations such as those found in c-Kit and FLT3 which have been shown to accelerate leukemogenesis. As relapse of inv(16) AML is invariably accompanied by increase of inv(16)+ positive cells, it is thought that relapse results from a failure of treatment to eradicate leukemia stem cells. In addition, the observation that a patient diagnosed with inv(16) AML later relapsed with an inv(16)+ pro-B cell leukemia, suggests that the inv(16) rearrangement had occurred in a stem cell/multipotent progenitor with reduced proliferation capacity (Boeckx, van der Velden et al. 2004).
Protein-Protein Interaction Inhibitors (PPIs).
Protein-protein interactions play a critical role in all aspects of signaling in the cell. In terms of their biological importance, these are highly attractive targets. However, until recently protein-protein interactions were widely considered to be undruggable, i.e., targets with a very low likelihood of success. This view was based on the large surface area and lack of curvature, i.e. pockets amenable to small molecule binding, frequently found at such protein interfaces. That view is rapidly changing as increasing numbers of such inhibitors are reported (Cochran 2000; Toogood 2002; Veselovsky, Ivanov et al. 2002; Gadek and Nicholas 2003; Fry 2006). Indeed, a recent review listed 17 targets for which such inhibitors have been developed (Gadek and Nicholas 2003).
Protein-protein interactions play a particularly important role in the regulation of transcription where the assembly of appropriate protein-protein complexes is essential for appropriate gene regulation. To that end, inhibition of protein-protein interactions involving transcription factors has substantial potential to alter gene expression and thereby the expression profile of cancerous cells (Arndt 2006). One recent success story in this regard is the development of inhibitors of the MDM2-p53 interaction. Binding of MDM2 to p53 leads to enhanced proteasome degradation of p53. Elevated levels of MDM2 are seen in a number of cancers. Roche initially developed a high potency inhibitor of this interaction (Vassilev, Vu et al. 2004) and others have developed additional inhibitors (Shangary, Qin et al. 2008). These inhibitors have been shown to abrogate the effects of reduced p53 dosage or mutant p53 by inhibiting this interaction thereby increasing the level of p53 in cells and enhancing p53 mediated apoptosis (Vassilev, Vu et al. 2004; Tovar, Rosinski et al. 2006; Efeyan, Ortega-Molina et al. 2007). These results demonstrate that targeting protein-protein interactions with small molecules is feasible and provide validation for our proposal to develop inhibitors of the CBFβ-SMMHC-RUNX interaction.
Targeted Therapy for Leukemia.
The general aim of targeted therapy against leukemia is to use our understanding of the cellular programs associated with the pathology of leukemia to design treatments with a markedly improved therapeutic index. The majority of such studies in the leukemia field have focused on targeting activated components of the cytokine receptor signaling pathway. One classic example in leukemia is the small molecule imatinib, which acts by targeting the ATP-pocket of the ABL kinase, thereby blocking the tyrosine kinase activity of BCR-ABL in chronic myelogenous leukemia (Druker 2004; Lydon and Druker 2004). The use of imatinib has improved CML treatment dramatically. Second generation inhibitors such as dasatinib are also efficacious against imatinib resistant CML. The application of this approach to other leukemias, however, has thus far been less fruitful. Clinical trials are now underway to test the therapeutic index of inhibitors for other components of cytokine receptor pathways compromised in leukemias, such as inhibitors of activated FLT3 receptor (CEP-701, MLN 518, and PKC 412) and JAK2 kinase, although it is unclear whether these inhibitors will show significant improvement in therapeutic index.
The identification of molecules that inhibit AML fusion oncogenes, thus abrogating the block in differentiation and inducing apoptosis, has thus far been lacking. Previous studies of targeted therapy reveal two aspects which are critical in the development of an effective drug: 1) the molecule should inhibit the oncogene function and induce cell differentiation or death with minimal alteration of normal hematopoietic progenitors and 2) the drug should effectively target the quiescent leukemia stem cells, not only the LSC-derived proliferating cells.
There is a long felt need in the art for compositions and methods useful for preventing and for treating acute myeloid leukemia, particularly involving the inv(16) fusion. The present invention satisfies these needs.